Eric K. Birks

Production of 20-HETE and Its Role in Autoregulation of Cerebral Blood Flow

In the brain, pressure-induced myogenic constriction of cerebral arteriolar muscle contributes to autoregulation of cerebral blood flow (CBF). This study examined the role of 20-HETE in autoregulation of CBF in anesthetized rats. The expression of P-450 4A protein and mRNA was localized in isolated cerebral arteriolar muscle of rat by immunocytochemistry and in situ hybridization. The results of reverse transcriptase-polymerase chain reaction studies revealed that rat cerebral microvessels express cytochrome P-450 4A1, 4A2, 4A3, and 4A8 isoforms, some of which catalyze the formation of 20-HETE from arachidonic acid. Cerebral arterial microsomes incubated with [(14)C]arachidonic acid produced 20-HETE. An elevation in transmural pressure from 20 to 140 mm Hg increased 20-HETE concentration by 6-fold in cerebral arteries as measured by gas chromatography/mass spectrometry. In vivo, inhibition of vascular 20-HETE formation with N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS), or its vasoconstrictor actions using 15-HETE or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), attenuated autoregulation of CBF to elevations of arterial pressure. In vitro application of DDMS, 15-HETE, or 20-HEDE eliminated pressure-induced constriction of rat middle cerebral arteries, and 20-HEDE and 15-HETE blocked the vasoconstriction action of 20-HETE. Taken together, these data suggest an important role for 20-HETE in the autoregulation of CBF.

Identification of a Putative Microvascular Oxygen Sensor

The vascular response to changes in oxygen levels in the blood and tissue is a highly adaptive physiological response that functions to match tissue oxygen supply to metabolic demand. Defining the cellular mechanisms that can sense physiologically relevant changes in PO2 and adjust vascular diameter are vital to our understanding of this process. A cytochrome P450 (P450) enzyme of the 4A family of omega-hydroxylases was localized in renal microvessels, renal cortex, and a striated muscle microvascular bed (cremaster) of the rat. In the presence of molecular oxygen, this P450 enzyme catalyzes formation of 20-HETE from arachidonic acid (AA). Prior studies have shown that 20-HETE potently contracts renal and cerebral arteries and arterioles. The present study demonstrates that 20-HETE constricts striated muscle arterioles as well. In both intact renal microvessels and enriched renal cortical microsomal enzyme preparations, the formation of 20-HETE was linearly dependent on PO2 between 20 and 140 mm Hg. Homogenates of cremaster tissue produced 20-oxygen HETE when incubated with AA. They also expressed message for P450 4A enzyme, as determined by Southern and Western blots. Administration of 17-octadecynoic acid (17-ODYA), which is a P450 4A inhibitor, attenuated the constriction of third-order cremasteric arterioles in response to elevation of superfusion solution PO2 from approximately equal to 3 to 5 mm Hg to approximately equal to 35 mm Hg. 17-ODYA had no effect on basal vascular tone or response of cremaster arterioles to vasoactive compounds. These results demonstrate the existence of P450 omega-hydroxylase activity and 20-HETE formation in the vasculature and parenchyma of at least two microvascular beds. Our data suggest that a P450 enzyme of the 4A family has the potential to function as an oxygen sensor in mammalian microcirculatory beds and to regulate arteriolar caliber by generating 20-HETE in an oxygen-dependent manner.

Cytochrome P450 Metabolites of Arachidonic Acid as Intracellular Signaling Molecules in Vascular Tissue

Recent studies from our laboratory have indicated that vascular smooth muscle cells (VSMC) metabolize arachidonic acid via a P4504A-dependent pathway to 20-hydroxyeicosatetraenoic acid (20-HETE), and that this system serves as a novel signal transduction pathway that plays a central role in the regulation of vascular tone. The major metabolite of arachidonic acid formed in cerebral and renal arteries is 20-HETE. The mRNA and protein for P4504A enzymes, which produce 20-HETE, have been localized in VSMC. 20-HETE is a potent vasoconstrictor, that acts in part by inhibition of the opening of the large conductance, calcium-activated potassium channel, and depolarizes VSMC membrane. A preliminary study also indicated that 20-HETE activates the L-type calcium current in cerebral arterial smooth muscle. Inhibition of the endogenous production of 20-HETE in renal and cerebral arterioles attenuates pressure-dependent myogenic tone in vitro, as well as autoregulation of renal and cerebral blood flow in vivo. There is also evidence that indicates that nitric oxide regulates the formation of 20-HETE by binding and inactivating the P450 heme moiety, thus providing a negative feedback control mechanism for this system. The data outlined suggest that 20-HETE could act as a intracellular second messenger that plays an integral role in the signal transduction processes underlying the development of pressure-dependent myogenic tone.

Role of P-450 Arachidonic Acid Epoxygenase in the Response of Cerebral Blood Flow to Glutamate in Rats

BACKGROUND AND PURPOSE Glutamate, a major excitatory neurotransmitter in the brain, has been implicated in the hyperemic response to increases in the activity of neurons, but the mechanism of glutamate-induced dilation of cerebral blood vessels is unknown. Glutamate has been shown to enhance the release of arachidonic acid (AA) in brain tissue and cultured astrocytes. We have previously shown that astrocytes metabolize AA to vasodilator products, epoxyeicostrienoic acids (EETs), and express a P-450 AA epoxygenase, P-450 2C11. We tested the hypothesis that glutamate-induced dilation of cerebral arterioles is mediated in part by changes in the formation and release of EETs by perivascular astrocytes. METHODS Primary astrocyte cultures were prepared from 3-day-old rat pups. The cells were labeled with [14C]AA, and the effect of glutamate on the formation of EETs from [14C]AA by cultured astrocytes was studied. The expression of P-450 2C11 protein in the microsomal fractions of cultured astrocytes was assessed by Western blot. In vivo cerebral blood flow measurements were made in adult rats by laser-Doppler flowmetry after administration of glutamate into the subdural space of the rat before and after treatment with miconazole. RESULTS Glutamate treatment (100 mumol/L for 30 minutes) induced a threefold increase in the formation of EETs from [14C]AA by cultured astrocytes, and the increase was inhibited by miconazole (20 mumol/L), an inhibitor of P-450 AA epoxygenase. Treatment with glutamate (100 mumol/L) for 12 hours increased the expression of P-450 2C11 protein in the microsomal fraction of cultured astrocytes. The response of laser-Doppler cerebral blood flow to administration of glutamate (500 mumol/L) into the subdural space of the rat was significantly attenuated after treatment with miconazole (20 mumol/L for 30 minutes). CONCLUSIONS These findings suggest a role for a P-450 AA epoxygenase in astrocytes in the coupling between the metabolic activity of neurons and regional blood flow in the brain.

Shear activated channels in cell-attached patches of cultured bovine aortic endothelial cells.

We investigated the response of inward rectifier K+ (IRK) currents in bovine aortic endothelial cells (BAECs) to shear stress. Shear evoked reversible hyperpolarization in current clamped BAECs. Voltage clamped BAECs exhibited large inward and small outward whole cell K+ currents blocked by cesium and increased in amplitude by exposure to shear stress. The open state probability of IRK channels in cell-attached membrane patches was increased within minutes of exposure to shear stress. IRK channels in inside-out patches were activated by increases in [Ca2+]i from 10−7 to 10−6 mM. We demonstrate that shear stress induces hyperpolarization and gating of single channel and whole cell IRK currents in BAECs.

Differentiation and identification of recombinant human erythropoietin and darbepoetin Alfa in equine plasma by LC-MS/MS for doping control.

Recombinant human erythropoietin (rhEPO) and darbepoetin alfa (DPO) are protein-based drugs for the treatment of anemia in humans by stimulating erythrocyte production. However, these agents are abused in human and equine sports due to their potential to enhance performance. This paper describes the first method for differentiation and identification of rhEPO and DPO in equine plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method comprised analyte extraction and enrichment by immunoaffinity separation with anti-rhEPO antibodies, dual digestion by trypsin and peptide-N-glycosidase F (PNGase F), and analysis by LC-MS/MS. Two unique deglycosylated tryptic peptides, (21)EAENITTGCAEHCSLNENITVPDTK (45) (T 5) from rhEPO and (77)GQALLVNSSQVNETLQLHVDK (97) (T 9) from DPO, were employed for differentiation and identification of rhEPO and DPO via LC retention times and major product ions. The limit of identification was 0.1 ng/mL for DPO and 0.2 ng/mL for rhEPO in equine plasma, and the limit of detection was 0.05 ng/mL for DPO and 0.1 ng/mL for rhEPO. Analyte carryover problem encountered was solved by adding 20% acetonitrile to the solvent of the sample digest to increase solubility of the peptides. This method was successfully applied to identification of DPO in plasma samples collected from a research horse following DPO administration and from racehorses out of competition in North America. Thus, it provides a powerful tool in the fight against blood doping with rhEPO and DPO in the horse racing industry.

Exercise-induced pulmonary hemorrhage

EIPH is a condition affecting virtually all horses during intense exercise worldwide. The hemorrhage originates from the pulmonary vasculature and is distributed predominantly bilaterally in the dorsocaudal lung lobes. As the condition progresses, the lung abnormalities extend cranially along the dorsal portions of the lung. An inflammatory response occurs in association with the hemorrhage and may contribute to the chronic sequela. Although conflicting opinions exist as to its affect on performance, it is a syndrome that is thought to increase in severity with age. The most commonly performed method to diagnose EIPH at the present time is endoscopy of the upper airway alone or in combination with tracheal wash analysis for the presence of erythrocytes and hemosiderophages. Because horses may not bleed to the same extent every time and the bleeding may originate from slightly different locations, these diagnostic procedures may not be extremely sensitive or quantitative. At this time, there is no treatment that is considered a panacea, and the currently allowed treatments have not proven to be effective in preventing EIPH. Future directions for therapeutic intervention may need to include limiting inflammatory responses to blood remaining within the lungs after EIPH.

A Modified Laryngoplasty Approach Promoting Ankylosis of the Cricoarytenoid Joint

Objective: To perform a modification to the standard laryngoplasty procedure in vivo that would result in ankylosis of the cricoarytenoid (CA) joint, and determine the stability provided to the abducted arytenoid in vitro. Study Design: Experimental study. Animals: Horses (n=8). Methods: Horses were assigned to either control laryngoplasty (n=3) or modified laryngoplasty (5) procedure. Endoscopic upper airway evaluations were used to measure right:left quotients 1 day and 3 months postoperatively to assess maintenance of abduction. Horses were euthanatized 3 months after surgery and larynges collected for measurement of translaryngeal impedance and histologic evaluation of CA joint ankylosis. Each specimen was exposed to increasing negative pressure with the sutures intact or cut while translaryngeal impedance was recorded. Data were analyzed using ANOVA with significance set at P<.05. Results: Loss of left arytenoid cartilage abduction at 3 months was greater in the control laryngoplasty group. Overall, impedance was significantly lower for the modified laryngoplasty group compared with the control laryngoplasty group and lower with the sutures intact than cut. Histologic evaluation of the joints confirmed fibrous bridging of the left CA joints of the modified laryngoplasty group. Conclusions: A modified laryngoplasty approach promotes ankylosis of the CA joint and decreases the loss of abduction of the arytenoid.OBJECTIVE To perform a modification to the standard laryngoplasty procedure in vivo that would result in ankylosis of the cricoarytenoid (CA) joint, and determine the stability provided to the abducted arytenoid in vitro. STUDY DESIGN Experimental study. ANIMALS Horses (n=8). METHODS Horses were assigned to either control laryngoplasty (n=3) or modified laryngoplasty (5) procedure. Endoscopic upper airway evaluations were used to measure right:left quotients 1 day and 3 months postoperatively to assess maintenance of abduction. Horses were euthanatized 3 months after surgery and larynges collected for measurement of translaryngeal impedance and histologic evaluation of CA joint ankylosis. Each specimen was exposed to increasing negative pressure with the sutures intact or cut while translaryngeal impedance was recorded. Data were analyzed using ANOVA with significance set at P<.05. RESULTS Loss of left arytenoid cartilage abduction at 3 months was greater in the control laryngoplasty group. Overall, impedance was significantly lower for the modified laryngoplasty group compared with the control laryngoplasty group and lower with the sutures intact than cut. Histologic evaluation of the joints confirmed fibrous bridging of the left CA joints of the modified laryngoplasty group. CONCLUSIONS A modified laryngoplasty approach promotes ankylosis of the CA joint and decreases the loss of abduction of the arytenoid.

Effects of chronic pulmonary overcirculation on pulmonary vasomotor tone.

BACKGROUND A model of shunt-induced pulmonary hypertension was used to study the effects of pulmonary overcirculation on endothelial nitric oxide synthase (eNOS) and cytochrome P450-4A (cP450-4A) vasodilatory mechanisms and related hemodynamic responses. METHODS An aortopulmonary shunt was constructed in 6-week-old piglets (n = 7, sham-operated controls n = 8). Hemodynamic measurements were made 4 weeks later under serial experimental conditions: baseline (fractional concentration of oxygen, 0.4); inhaled nitric oxide, 25 ppm (INO); hypoxia (fractional concentration of oxygen, 0.14); hypoxia + INO; N(omega)-nitro-L-arginine methylester (L-NAME 30 mg/kg intravenously, competitive NOS inhibitor); and L-NAME + INO. Lung protein levels of eNOS and cP450-4A and NOS activity were compared between groups. RESULTS Shunted animals had a higher baseline pulmonary artery pressure (p < 0.05). L-NAME resulted in a greater increase in pulmonary vascular resistance in shunted animals (150% +/- 26% shunt versus 69% +/- 14% control; p = 0.01). The INO administered during baseline conditions decreased pulmonary vascular resistance only in control animals (p < 0.05). Protein levels of eNOS and NOS activity were similar in both groups; however, cP450-4A protein levels were decreased in the shunted group (p = 0.02). CONCLUSIONS The NO production was preserved in shunted animals but they demonstrated greater vasodilatory dependence on NO, evidenced by an exaggerated increase in pulmonary vascular resistance after NOS inhibition. Loss of the cP450-4A vasodilatory system may be the driving force for NO dependency in the shunted pulmonary circulation.

The effects of frusemide on racing times of Standardbred pacers

Seven hundred and eighty-eight Standardbred pacers competing in 8378 races at one racetrack were analysed to determine the effects of the administration of prerace frusemide on racing times (RT). Frusemide was administered i.v. 4 h before the race to pacers diagnosed with exercise-induced pulmonary haemorrhage (EIPH). Of the pacers, starting in the 1997 racing season, 32.5% received prerace frusemide. This study demonstrated that administration of frusemide prior to racing significantly decreased RT. There was an overall significant decrease (P<0.00001) in RT of 0.67 s. The overall RT for horses, geldings, and females, were mean +/- s.e 117.91 +/- 0.06, 118.20 +/- 0.03 and 118.86 +/- 0.04, respectively. RT progressively decreased until age 6 and increased thereafter. Horses, geldings and females ran a mean of 0.46, 0.31 and 0.74 s faster, respectively, with prerace administration of frusemide. This decrease in RT following prerace administration was most pronounced in younger pacers. In this study, a greater percentage of older pacers received prerace frusemide; however, the effect of frusemide on RT was decreasing with age. Prerace venous acid-base screening was performed in 2729 of the pacers competing. Pennsylvania Harness Racing Commission Regulations disqualify Standardbreds from racing with a base excess of over 10 and 12 mmol/l for Standardbreds without and with prerace administration of frusemide. The prerace venous acid-base levels were not significantly related to RT and, for those Standardbreds also sampled following the race, there was no correlation between pre- and postrace acid-base status.